A lot of G-protein-coupled receptors (GPCR) trigger, upon binding of an agonist, a transient increase in intracellular calcium concentration. This variation acts as an internal secondary messenger and is an important modulator of many physiological mechanisms (reviewed by Rink (1990), Tsunoda (1993) and by Santella & Carafoli (1997)). Measurement of intracellular calcium concentration in cells expressing a GPCR can thus be used to monitor the efficacy of activation of a GPCR by various compounds known —or suspected—to be a ligand for this GPCR.
The activation of other receptors such as ions channels may also induce an intracellular calcium concentration.
Changes in calcium concentration can be detected by several means and methods, like the use of fluorescent dyes (for example: fura-2, fluo-3, fluo-4 and indo-1).
However, Ca++sensitive dyes have limitations. Activation of the dyes with an excitation beam requires complicated and expensive instruments and limits the use of the plastic labware such as microtiter plates.
Another method for intracellular calcium concentration measurement is the use of cell lines overexpressing a GP and apoaequorin, such an described by Sheu et al. (1993). In this system, cells expressing apoaequorin are incubated with coelenterazine, which is the co-factor of aequorin. During this incubation, coelenterazine enters the cell and conjugates with apoaequorin to form aequorin, which is the active form of the enzyme. Upon incubation of the cells with an agonist of the GPCR, intracellular calcium concentration increases. This increase leads to the activation of the catalytic activity of aequorin, which oxidises coelenterazine and yields apoaequorin, coelenteramide, CO2 and light. Once the photon has been emitted, the complex must dissociate and apoaequorin must recombine with a new coelenterazine molecule to be able to emit light again. Thus, in this system, measurement of light emission following agonist addition reflects its ability to activate the GPCR and thus to increase intracellular calcium concentration. Because light is emitted only during 20 to 30 seconds after; activation of the GPCR, recording of the emitted light must be performed during the few seconds following agonist addition to the cells. This flash-type signal is due to the fact that (1) intracellular calcium increase triggered by GPCR is only transient and (2) as mentioned earlier, after oxidation of coelenterazine, apoaequorin must recombine with coelenterazine to be able to emit light again.
The Patent Application EP-0341477 teaches the expression of jellyfish photoprotein aequorin in a mammalian cell system by cloning gene pAQ440 specifying the biosynthesis of the aequorin into an expression vector plasmid of a mammalian cell system, subjecting the resulting plasmid to transfection and producing the photoprotein aequorin in the mammalian cell.
The U.S. Pat. No. 5,422,266 describes a gene encoding apoaequorin protein included in a vector capable of expressing the apoaequorin in E. coli. 
The U.S. Pat. No. 5,714,666 describes mammalian cell lines or transgenic animals expressing apoaequorin and a receptor involved in the modulation of intracellular calcium. This document also describes a method of measuring intracellular calcium comprising adding coelenterazine cofactors to said mammalian cells expressing apoaequorin and measuring photoemission where emission of photons is indicative of intracellular calcium concentration.
However, the methods of the state of the art require firstly the spreading of cells from a mammalian cell line expressing apoaequorin on a solid support (for example a 96-well plate), secondly the addition of the coelenterazine cofactor upon the cells and incubation to, reconstitute a functional aequorin, thirdly the preparation of the agent affecting a receptor involved in the modulation of intracellular calcium concentration, and its addition to the prepared cells, and finally the measurement of the photoemission.
Furthermore, as mentioned above, light is emitted only during 20 to 30 seconds after activation of the GPCR. Therefore, the recording of the emitted light must be performed during the few seconds following agonist addition to the cells.
Therefore, the methods used in the state of the art are not adequate for a detection based upon high-throughput screening level, which usually need luminometer(s) and require the use of microtiter plates for the testing of thousands of compounds.